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Lab Report: Lab Rotations (Spring 2007) in the laboratory of Prof. Dr.

Angela Koehler
under the supervision of Dr. Katja Broeg and Sonja Einsporn, PhD;,
Alfred Wegener Institute for Polar and Marine Research (AWI)
Am Handelshafen 12, 27570 Bremerhaven, Germany

IMPACT OF METAL POLLUTION IN LIVER TISSUES OF


CORKWING WRASSE FISH (SYMPHODUS MELOPS L.) AT
CELLULAR LEVEL

By
Kedar Ghimire
Jacobs University Bremen
Lab rotations III, IV in AWI k.ghimire@jacobs-university.de 2

Structural differences in organelles and its consequences in the liver


tissues of Corkwing Wrasse fish (Symphodus melops L.) sampled
from differently polluted coastal sites of Norway

Kedar Ghimire,
Jacobs University Bremen, School of Engineering and Science, Campus Ring 1, 28759 Bremen,
Germany

Wrasse (Symphodus melops L.) is an important marine species for monitoring the
environmental and health effects of contamination in North Sea. Due to the toxic
substances like PAH (polycyclic aromatic hydrocarbons), biocide(C-Treat 6), TBT etc
released by aluminium smelters; metal contamination of coastal water due to copper
mines; the habitats of this fish have been negatively effected. Many of these fishes have
been found to be effected with various diseases that directly affects the vital metabolic
organs of the body like the liver hinting to the fact that the situation of life forms in these
areas are in peril. Through this study, we have attempted to explore the liver tissues
(hepatocytes) from various wrasse samples living in metal (copper) contaminated sites
and reference sites and make a comparable analysis of the structural and functional
changes observed in the cell organelles at electron microscope level. We conclude that Cu
contamination is harmful and it affects the cell organelles in liver tissues of Wrasse in
different ways.

Keywords: Hepatocytes, lipid, copper, metallic crystals, metabolism, glycogen, electron


microscopy
Abbreviations: TBT: Tributyltin
PAH: Polycyclic aromatic hydrocarbon
EM: Electron microscopy

Introduction:
Wrasse is an interesting fish species functions. Liver cells secrete bile which
whose gender changes from female to male emulsifies fat and helps change the acidic pH
during the life time (a protogyn) (Broeg et al, of stomach into neutral pH of the intestine. Bile
2007). It has a flat body structure. Specific collects in the bile capillaries, which then unite,
chemical impacts are expected to change forming bile ducts. The bile canaliculus is a
morphology and consequently, the function of structure formed by grooves on the contact
its organs. Increasing frequencies of surface of adjacent liver cells, i.e. the dilated
toxipathic lesions and liver tumors have been intercellular space between adjacent
reported in other fish from areas with chemical hepatocytes. Bile forms in these canaliculi and
impact of pollution (Gardner et al., 1991; then flows into small ducts, and finally into
Koehler et al., 1992; Johnson et al., 1993; larger hepatic ducts.
Stein et al., 1990; Stentiford et al., 2003). We
fear Wrasse can be another such victim. Figs. 1.2 and 1.1 in the next pages
show a liver tissue with a normal nucleus,
Fish are poikilothermic vertebrates so plenty of glycogen granules, lot of vesicles,
they change their metabolism according to the lysosome and plenty of mitochondria. It should
temperature variations throughout the year be noted that the liver is the major site for Cu
and all those changes are reflected in the liver. excretion (in the bile) in vertebrates. While
Fish are highly susceptible to environmental copper is an endocrine disrupter in the aquatic
variations and respond sensitively to pollutants animals and has a number of neuro-endocrine
than other various mammals (Munsi and effects in vertebrates (Handy, 2003).
Dutta, fish morphology, 1996). The liver of the
wrasse has many digestive and storage The fish were sampled from five
different fjord sites in Norway. Site 1 was
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SalvØy, considered to be an outer reference of 3. The sections were then washed with dist.
site on the west side of Karmoy. Site 2 was water and dipped in ethanol for dehydration
Visnes- a highly copper and zinc contaminated and quickly taken out and dried. After light
site on the west side of Karmoy. In this site, microscopic analysis, it was found that
both tailings and slag was dumped too. Site 3 samples heated at 2 and stained for 2 minutes
was FØrlandsfjorden- an extremely sheltered produced better results and were
fjord representing the inner part of the fjord subsequently used.
system, with small boat traffic and some small
farms that drain to the fjord with vast amount After light microscopy, the
of mussels found along the shores of the fjord. block sections were marked after considering
Site 4 was Bokn- a reference site in the their special characteristics to observe under
exposed part of the fjord system. Site 5 was EM. These marked sections were prepared in
HØgevarde- a site just north of the PAH block removing other unnecessary areas with
discharge from the Alumina smelter in blade. Then the microtome (Model Leica EM
Karmsund. Other discharge there consisted of UC6) was used to prepare ultra sections for
biocide, TBT. Among these sites, our study EM. The settings of 1 mm/s speed and 60
focused on site 2 and 3. Site 2 was influenced feed/nm was used for this purpose. The sliced
by an old copper mine which was in sections were placed in small grids carefully.
production 1865-94 and a new production for Then staining was performed. First, the
a few years until 1965. The area closest to the sample was stained with Uranyl acetate for 5
old mine had no sign of life. The sea water minutes, and then washed thoroughly with
was exposed to the metals mainly copper and ddH2O. Then the sample was again stained in
zinc from the fillings and the run off from land. lead citrate for exactly 1 minute and washed
Station 3 was our reference site. thoroughly well and let to dry for a night. Few
We used the methods of microscopic equivalent samples were not stained so that
analysis at light and EM level in our study to comparable analysis could be done between
observe structural changes in the liver tissues stained (contrasted) and unstained
and the consequences of these changes (uncontrasted) sections of the same region.
towards the physiology and adaptation of
Wrasse. Results:
We tried to see and note the
Materials and Methods: differences in structure and consequently in
Our samples were embedded in the function of cellular organelles of Corkwing
year September 2001 and were preserved wrasse fish from metal sites and reference
safely. site. The differences between normal and
For electron microscopy, after pollution-effected tissues will be discussed in
embedding liver tissues into epoxy resin, a full detail in the discussion. Our results could
microtome (Model Leica EM UC6) equipped be explained through various EM images of
with a diamond knife was used to cut first very the liver tissue of Corkwing Wrasse fish.
thin sections for examining under light Overview
microscope (Zeiss Axioskop). The settings of
microtome was speed (mm/s) =1 and
feed/nm=500. For this, three samples (Nr. 2,
3, 4) from station 2 and two samples (Nr. 1, 5)
from station 1 were chosen. Five slides from
each sample were prepared. Among them,
two of each sample were stained with Toluene
Blue 0.5% in Na2CO3. Toluene blue was
filtered and the tissue sections were stained
for 1-2 minutes in it. We had used variations
for this process. Two slides for each sample
were prepared. One sample was heated
(Stuart SB 300 heater) to magnitude 2 and
was stained for 2 minutes. The other sample
Fig 1. Transmission EM Overview of the tissue
was stained for 1 minute with heat magnitude from station 2 (polluted site) at 3000 × magnification
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Fig 1.1 Transmission EM Overview of the liver Fig3. Transmission EM of a mitochondrial overview
tissue from station 3 (reference site) at 12000 × in section of liver tissues from station 3 at 20000×
magnification magnification

Figs. 4, 4.1, 5, 6, 7, 8, 9, 10 under the same


heading as fig. 2 could be found in appendix 1.
Observations on mitochondria and
lysosome Observations on nucleus and
Endoplasmic Reticulum

Fig 2. Normal mitochondria seen in liver tissues Fig 11. EM of Rough endoplasmic reticulum from
from station 3 at 12000 × magnification station 2 at 20000 × magnification
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Observations on metal deposits in


tissues and bile canaliculus

Fig 12. Assumed metal deposits inside the cells Fig 18. Black deposits in intracellular space at
from station 2 at 7000× magnification 20000× from station 2.

The black deposits in figure 12 are assumed Figs. 13, 15, 16, 16.1, 17 and 21 under the
to be pieces of metallic elements. It was seen same heading as fig. 12 could be found in
at random places and not uniformly. appendix 1.

Fig 14. Bile canaliculi with black deposits in the Fig19. Assumed metal deposits in Bile canaliculus
surrounding along intracellular pathways at 4400 × with lipids alongside at 3000× magnification,
magnification from the station 2 metal site. (station 2- nC)
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Discussion:
Aqueous Cu has been reported to
accumulate in several tissues like gill, kidney
and liver during chronic exposure and there is
lesser accumulation in muscle (Handy, 2003).
The metal site at Visnes is a chronic exposure
to fish since there is an abandoned mine. In
such type of exposure, it had been found fish
have more time to down regulate Cu uptake
through the gills and distribute newly acquired
copper to the liver for excretion to minimize
the toxological effects of copper (Grosell et al.,
1996, 1997, 1998). It is also known that fish try
to adjust to the metal exposure by initiating
complex physiological adjustments like
increased oxygen consumption, increasing
neutrophils, altered immunity, increasing ionic
regulation and altered cellularity (Handy,
2003). Copper demonstrates a high affinity for
Fig 20. Black deposits in the intercellular space thiol groups and is therefore capable of
near bile canaliculi at 3000× magnification- non- severely disrupting many metabolic functions
contrasted image (station 3)
in the cell (Hultberg et al., 1998).

Fig 1.2 (see Appendix 1) is a


Observations on lipid and glycogen transmission electron micrograph which shows
present in tissues a clear overview of the wrasse liver tissue at
3000× magnification. The section was from
station 3 which consisted of our reference site.
Lysosome engulfing a lipid molecule could be
seen. A normal nucleus with general lipid
droplets was seen. No abnormalities were
seen in the cells from the liver tissues of
Wrasse from reference site as expected. Fig
1.1 shows another section from the station 3
at higher magnification of 12000×. A
noticeable observation was that lots of
vesicles could be seen, implicates that the cell
was quite active with all types of intra-cellular
activities going on and consequently, should
be a very healthy cell. Mitochondria seemed to
be coupled with the lipid droplet.

Compared to cells from reference site,


fig 1 shows EM of the liver tissue from site 2-
metal contaminated sites. No. of glycogen
Fig 22. Glycogen penetrating lipid and granules was much higher than those seen in
mitochondria attached to lipid from liver the reference site. It can be inferred that there
tissues at station 2 at 30000× magnification is at least some disruption in gluconeogenesis
due to which glycogen couldn’t convert
Fig 23, 24, 25 and 26 under the same heading sufficiently into glucose. Carattino et al. (2004)
as fig. 22 could be found on the appendix 1. had shown that Cu significantly inhibits
glucose-6-phosphate dehydrogenase activity
in-vitro in their study on effects of Cu on
metabolism through pentose phosphate
pathway in toad ovaries. Cu has also been
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found to interfere with the glycolytic pathway Figure 4.1 (reference site) shows the
(Strydom et al., 2006). Glycogen granules presence of lipid alongside mitochondria. The
were seen attached to the periphery of the lipids also were seen to be degraded by
lipid molecules. The reason for this close lysosome. Figure 5 shows a non contrasted
association could be that glycogen as a image of a cluster of lipids attached to
polymer of glucose can form covalent bonds mitochondria, this combination of mitochondria
with the fatty acid chains in lipid. Fatty acids and lipid structure hints towards a possible
are made by repeatedly joining together the interaction between mitochondria and lipid for
two-carbon fragments found in acetyl-CoA and degradation of lipids. Eugene P. Kennedy and
then reducing the (-CO-) part of the molecule Albert Lehninger had already demonstrated in
to (-CH2-). In this way, the hydrocarbon chain 1948 that enzymes of fatty acid oxidation in
can become the hydrophobic, energy storing animal cells are located in the mitochondrial
part of the fatty acid. matrix. It is known that A-Methylacyl-CoA
The number of lipid molecules is racemase, found in both mitochondria and
considerably high in comparison to cells from peroxisomes, is required for the metabolism of
reference site. This clearly hints towards some isoprenoid compounds, e.g. cholesterol to bile
negative effects on the reactions catalyzing acids, and other methyl branched lipids. So,
lipid break down. In reverse, there is increased this could well be happening in case of fish as
importance of the catabolism of lipids in such well. Mitochondria could be well affected due
cells so that they wouldn’t accumulate to to metal deposits and thus might not be
harmful level. Lipid peroxidation in response to functioning properly to produce enzymes for
copper exposure has been reported in lipid oxidation. Also in the same image, faults
freshwater crab (Vosloo et al, 2002). In our and cuts on lipids could be seen where metal
images, lysosome was seen attached to the crystals had aggregated. This could be seen
lipid and could be in the process of degrading as the harsh physical effects of metal crystals
lipids. Lipid peroxidation is considered as a on the cellular organelles. Definitely, metal
measure of oxidative stress and general stress precipitates seem to affect the cell organelles
thus is an indicator of fish health as a whole chemically as well as physically.
(Marcogliese et al, 2005). A suspected
transport of black deposits (possibly metal Figure 6 shows glycogen filled
crystals) was seen at 3000 M denoted in the lysosome and high amount of glycogen all
figure as SP. An increased stimulation of ROS around. It shows that there has been
production by metals may lead to an obstruction in the pathway of conversion of
imbalanced oxidative stress condition in fish glycogen. The cell seems to be very active as
that may result in physiological alterations a lot of lysosome and vesicles were seen.
(Sies, 1993, Paris-Palacios et al., 2000 and Again, an elongated and irregularly shaped
Varanka et al., 2001). Huge lipids were seen mitochondria could be seen in the figure. In
in sections from polluted sites. figure 7, lysosome degrading lipid and a lot of
black metal crystals were seen. However, in
Figure 2 and figure 3 shows an image fig. 6, lysosome seemed to have weaker
of the tissue at 12000 M from station 3, membrane structure since the membrane
showing normal mitochondria (1-5 µm) with lining looked very irregular compared to
parallel cristae. There was no sign of any normal lysosome. It had been already shown
precipitates in these mitochondria. Lysosome that the lysosomal membrane stability of
seemed to be in pearl structure and ER was wrasse was impaired at the sites of PAHs and
dilated. Again, a lot of vesicular activity was organic contamination (Einsporn and Koehler,
seen around. In contrast to these, EM of liver 2007) so it seems metallic pollution results in
tissues from polluted sites (fig 4) showed same, but could be in lesser extent as PAH
elongated mitochondrias, which seem to be and organic contamination are harsh and
damaged and totally irregular in shape. A more effective than simple metals like copper.
considerable amount of precipitate was seen The interior of the lysosomes (pH 4.8) is more
inside the mitochondria. The parallel cristae acidic than the cytosol (pH 7). The lysosome
structure wasn’t seen but cristae seemed to be single membrane stabilizes the low pH by
+
damaged at various places in the mitochondria pumping in H from the cytosol, and also
as it was seen at random positions within the protects the cytosol and the rest of the cell,
mitochondria.
Lab rotations III, IV in AWI k.ghimire@jacobs-university.de 8

from the degradative enzymes within the mitochondria are sensitive to cellular stress
lysosome. and have a pivotal role in the initiation of
programmed cell death. It could be proposed
Fig. 8 clearly shows glycogen and that the structural change in the mitochondria
ribosome, ribosome attached to endoplasmic begins with the degradation of cristae due to
reticulum. Glycogen was seen to be of two metal pollution. In fig. 12 and fig. 14, dense
types. Both bigger than ribosome in size, and metal deposits could be seen near bile
were stained differently. One was darkly canaliculi. The tissue sections were from
stained electron dense granules while the copper polluted site. It is of specific
other was lightly stained. Glycogen appeared observation that the density of the black
as electron-dense rosettes, termed alpha deposits is around bile canaliculi and metal
particles. However, glycogen-rich and crystals could be seen attached to the grooves
glycogen-poor liver is differentiated by no. of of bile canaliculus. This could well be the
alpha particles rather than in their size. export and elimination pathway for these
precipitates from the fish through bile
It is noticeable that some particles of canaliculi. There is less possibility for these
glycogen seem to have combination of both deposits to be background staining or dirt
light and dark areas. This could either be due particles because it can be seen clearly that it
to complex formation of glycogen with is not uniform throughout the tissue and
ribosome or it is also known that alpha localized in certain specific areas.
glycogen contains beta particles within itself.
And the alpha particles additionally contain If it had been the remains of staining
various enzymatic proteins involved in the procedure, it should well have been seen all
synthesis of glycogen and hence they are around the tissue. Also, the sections were
called glycosomes (Rybicka, 1996). The dead so there is no reason that the dirt, if it
shape of black particles in this reference site was, should be attached to the grooves of bile
image seemed to be different from an canaliculus instead of any other parts of
uncontrasted image from metal site (fig. 26). It tissue. But if these were metal deposits, it
is hard to distinguish between ribosomes and makes perfect sense they were being
glycosomes because both types of organelles eliminated while the samples were taken and
appear in U/Pb stained sections, as 20-30 nm prepared for EM in 2001 and remained there
particles. Also both could attach to ER frozen. Fig. 15 (see appendix I) shows the
membranes and cytoskeletal components same at 12000×. Our evidence is strongly
(Hesketh and Pryme, 1991). supported by Fig. 16 (see appendix I),
16.1(see appendix I) and 20 and 21(see
Fig. 9 shows an uncontrasted image appendix I). These were not stained before
of lysosome degrading lipid at 12000×. Fig. 10 electron microscopy and were noncontrasted
(12000×) shows bulged mitochondria in images but they still showed black particles on
contrast to the normal mitochondria in liver the canaliculus and intercellular space. Also
tissues of reference site (Fig 2, 12000×). A evident was the fact that the cells in direct
huge difference in size could be seen between contact with bile canaliculus showed more
mitochondrias from polluted and unpolluted particles than the secondary cells after them
sites. In polluted site, mitochondria were and the particles were polarized at one side of
considerably swelled with cristae at random bile canaliculus, (see figure 12, 14, 15). In fig.
positions. Again, we saw certain amount of 13 (see Appendix I); unlike in the middle of the
granules like precipitates in the mitochondria. cell, the lining of the tissue was filled with
We propose the swelling of mitochondria is black deposits. It could be assumed that these
directly related to the effect of precipitates on were metal crystals but it couldn’t be
the mitochondria. confirmed whether it is copper or the residues
of uranyl citrate and lead acetate, since this
In figure 11, the mitochondria seemed was a contrasted image stained with these two
to be damaged with almost no cristae. The compounds. Numerous lipid droplets were
mitochondria are greatly enlarged and are seen and this section was from polluted site.
filled with concentric cristae, which is
abnormal. Also, the mitochondrial matrix Fig. 17 (see appendix I) and 18 shows
appeared to be inexistent. It is well known that black deposits passing through an intercellular
Lab rotations III, IV in AWI k.ghimire@jacobs-university.de 9

space presumably. When closely observed, especially copper. Very important organelles
the black deposits are minute rectangular like the mitochondria which are the power
shaped crystalline structures with sharp house of cells were found to have irregular
edges. These prove that they are metallic structures, excess of glycogen was seen and
crystals. Presence of abnormally accumulated amount of lipid droplets were by far, in lot
metal crystals show detoxification function of more amount than a normal cell should have.
liver has been impaired.
On the other hand, due to the small
Fig. 22 shows us the glycogen scale and time of this lab study, many
enclosed with lipid. Both dark and light observations could not be decided
granules of glycogen can be seen. It was conclusively. In future, Autometallography
interesting to see mitochondria blocking the should be performed at the light and electron
way for glycogen to come out of the lipid microscope levels to provide information on
droplet. It might well be that this is one of the the intracellular distribution of metals as well
ways through which glycogen make their way as evidence of different responses to metal
inside lipid and are later trapped. Similar case accumulation. By checking the pH in and
was seen in fig 24. Mitochondria squeezed around lysosome, it could be found out
between lipids, limiting the movement of whether they had weak membrane structure in
glycogen. The occurrence of glycogen real since a considerable variation from pH 4.8
inclusions in the liver hints that the metal means the proton pumps and chloride ion
deposits could be causing biochemical stress channels on the membrane to maintain the pH
in the fish from polluted areas. The is not functioning well, which is related to
accumulation of glycogen in hepatocytes could damages in membrane of lysosome. The
result in type IV glycogen storage disease effects of structural differences in mitochondria
(amylopectinosis) (Sherlock and Dooley, 1997; should be studied thoroughly and quantitative
Peplow and Edmunds, 2005). analysis should be done on the unknown
effects on ATP production due to loss of
Fig. 23 is a wonderful case showing cristae seen on affected mitochondria.
the two types of glycogen, dark and lighter in
color complexion. On liver, glycogen appears
as electron dense rosettes, which are alpha Acknowledgements
particles so we assumed the darker particles I would like to thank Ute Marx
to be alpha glycogen. The structural backbone (Alfred Wegener Institute) for her technical
of ER could be seen clearly with ribosomes. It assistance in electron microscopy and Sonja
could be inferred that glycogen particles could Einsporn, PhD. and Dr. Katja Broeg for their
have also formed a stable complex with guidance and supervision during the
ribosomes. experiments.

Figures 25 and 26 show lipid droplets


in tissues from metal site at 12000 ×. Also References:
evident are the lipids being degraded by
lysosome selectively. Generally, Lysosomes Broeg, K., Kaiser, W., Bahns, S., Koehler, A.(2007)The
were seen to degrade lipids with black metal liver of wrasse-morphology and function as a mirror of
deposits before others. In Cu-loaded animals point source chemical impact. Marine environmental
there is overburden on biliary excretion research. 14th international symposium pollutant
responsed in marine organisms. May,6-9 2007, Brazil
pathways. It is typified by the sub-cellular
localization of Cu in non-cytosolic fractions, CARATTINO MD, PERALTA S, PÉREZ-COLL C, NAAB
especially lysosomes (Klaverkamp et al., F, BURLÓN A, KREINER AJ, PRELLER AF and
1991). This is clearly seen in fig 26, which is FONOVICH DE SCHROEDER TM (2004) Effects of long-
term exposure to Cu2+ and Cd2+ on the pentose phosphate
an uncontrasted image of wrasse liver pathway dehydrogenase activities in the ovary of adult
exposed to metal site. Bufo arenarum: Possible role as biomarker for Cu2+
toxicity. Ecotoxicol. Environ. Saf. 57 311-318
To summarize, the study was
C Strydom, C Robinson, E Pretorius, JM Whitcutt, J
successful to illustrate that various cell Marx, and MS Bornman (2006) The effect of selected
organelles could be affected due to physical metals on the central metabolic pathways in biology: A
and chemical toxicity of metal pollution review. ISSN 0378-4738 = Water SA Vol. 32 No. 4
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metal pollution controls in a smelter by using


Einsporn S. and Koehler A., (2007) Lysosomal changes metallothionein and other biochemical responses in fish.
in Wrasse and in blue mussel from differently polluted In: M.C. Newman and A.W. McIntosh, Editors, Metal
norwegian fjord sites (2007), Ecotoxicology Ecotoxicology—Concepts and Applications, Lewis
Publishers Ltd., Chelsea, MI, USA (1991), pp. 33–64
Grosell, M.H., Boëtius, I., Hansen, J.M. and
Rosenkilde, P., 1996. Influence of pre-exposure to sub- Marcogliese DJ, Brambilla LG, Gagne F, Gendron AD
lethal levels of copper on 64Cu uptake and distribution (2005) Joint effects of parasitism and pollution on
among tissues of the European eel (Anguilla anguilla). oxidative stress biomarkers in yellow perch Perca
Comp. Biochem. Physiol. Part C 114, pp. 229–235 flavescens. Diseases of aquatic organisms. Vol 63: 77-84

Grosell, M.H., Hogstrand, C. and Wood, C.M., 1997. Munshi J.S., Dutta H.M. (1996)
Copper uptake and turnover in both Cu acclimated and Fish Morphology: Horizon of New Research, CRC press
non-acclimated rainbow trout (Oncorhynchus mykiss).
Aquat. Toxicol. 38, pp. 257–276. Peplow D, R Edmonds, 2005. The effects of mine waste
contamination at multiple levels of biological organization.
M.H., Hogstrand, C. and Wood, C.M., 1998. Renal Cu Ecological Engineering, 2005 (Vol. 24) (No. 1/2)
and Na excretion and hepatic Cu metabolism in both Cu
acclimated and non acclimated rainbow trout Rybicka KK (1996) Glycosomes- the organelles of
(Oncorhynchus mykiss). Aquat. Toxicol. 40, pp. 275–291. glycogen metabolism. Tissire Cell 28:253

Sherlock S. and Dooley J.S. Diseases of the Liver and


Handy D. R. (2003) Chronic effects of copper exposure Biliary System, 10th ed. Blackwell science, Oxford
versus endocrine toxicity: two sides of the same
toxicological process? CBP. Part A 135 25-38. Sies H. (1994) Oxidative stress: oxidants and
antioxidants, Experimental Physiology 82.2 pp 291-295
HULTBERG B, ANDERSSON A and ISAKSSON A
(1998) Alterations of thiol metabolism in human cell lines
induced by low amounts of copper, mercury or cadmium Vosloo A., Aardt van W.J., Mienie, L.J. (2002) Sublethal
ions. Toxicol. 126 203-212. effects of copper on the freshwater crab Potamonautes.
Comp. Biochem and Physiology 695-702
J E Hesketh and I F Pryme (1991) Interaction between
mRNA, ribosomes and the cytoskeleton. Biochem. J. 277 (1–0)

J.F. Klaverkamp, M.D. Dutton, H.S. Majewski, R.V.


Hunt and L.J. Wesson, Evaluating the effectiveness of
Lab rotations III, IV in AWI k.ghimire@jacobs-university.de 11

APPENDIX 1, Lab rotation III and IV

Figures included in the discussion

Fig 15. Bile canaliculi with black crystal deposits


Fig 1.2 Overview of the liver tissue from station 3 at 12000× magnification from metal site at station
(reference site) at 3000 × magnification 2.

Fig 13. The bile duct with a black metal lining Fig 16. Bile canaliculi with black crystal deposits
throughout edges at 3000× magnification, from at 20000× magnification from station 2
metal site. (uncontrasted image)
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Fig 16.1. Metal deposits in the bile canaliculi at


Fig 21. Assumed metal crystals from station 2 at
12000× magnification from station 2
12000× magnification (non contrasted image)
(uncontrasted image)

Fig 17. Black deposits along an intracellular Fig 23. Glycogen around lipid and ER at 12000 ×
space at 12000 × magnification from station 2 magnification (station 2)
L represents lipid while G shows glycogen
granules.
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Fig 24. Glycogen around lipid and at 20000 × Fig26. Lipid filled with metal crystals being
magnification (station 2) degraded by lysosome at 12000×
magnification from station 2 (non contrasted
image)

Fig 25. Lipids being degraded by lysosome in Fig 4. EM of Elongated mitochondria from station
liver tissues at 12000× magnification from station 2 at 12000×.
2 (uncontrasted image- metal site)
Fig. 4 shows a section of liver of Wrasse
which was collected from station 2- the
metal polluted sites. Unusually long,
stretched mitochondria were found. A lot of
glycogen was seen at most of the places.
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Fig 6. Mitochondria and lysosome from station 2


Fig 4.1. Lysosome in liver tissues degrading
at 12000×
lipids from station 3 at 12000 ×

Fig 5. EM of Mitochondria in an uncontrasted Fig7. Lysosome filled with black deposits from
image, from station 2 at magnification 12000× station 2.
magnification

Mitochondria can be seen squeezed


between lipid droplets. Faults on lipids could
also be seen where black deposits had
accumulated.
Lab rotations III, IV in AWI k.ghimire@jacobs-university.de 15

Fig 8. Ribosome, glycogen from liver tissues Fig 10. An uncontrasted image of mitochondria
from station 3 at 20000 × magnification and lipid in liver tissues from metal site at 12000
× magnification

Fig9. Lysosome degrading lipid in uncontrasted


TM image from station 3 at 12000 × magnification

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